Production, composition and toxicology studies of Enzogenol® Pinus radiata bark extract.

Enzogenol® pine bark extract is a dietary supplement and food ingredient produced by water extraction of Pinus radiata. We present production method, composition, and safety data from rat and dog toxicological and human clinical studies. The dry powder contains proanthocyanidins (>80%), taxifolin (1-2%), other flavonoids and phenolic acids (up to 8%), and carbohydrates (5-10%). Reverse mutation assays showed lack of mutagenic activity. Single and 14-day repeat dosing in rats and dogs had no influence on body weight, feed consumption, blood chemistry, and haematology at any dose level. There were no treatment related findings on gross and detailed necroscopy, organ weights, organ weight ratios and histology. The only adverse events were emesis and diarrhoea in dogs occurring mainly in un-fed condition and at the highest dose level in a total of 18% of applications. The MTD and NOAEL in the present rat and dog studies were 2500 and 750 mg/kg/day, respectively. Consumption of 480 mg/day for 6 months and 960 mg/day for 5 weeks in two human studies showed Enzogenol® had no adverse influence on liver and kidney function, haematology, and did not cause any adverse events. Our studies indicate lack of toxicity of Enzogenol® and support safe use as a food ingredient.

Expression of IFITM1 in chronic myeloid leukemia patients.

We investigated the peripheral blood gene expression profile of interferon induced transmembrane protein 1 (IFITM1) in sixty chronic myeloid leukemia (CML) patients classified according to new prognostic score (NPS). IFITM1 is a component of a multimeric complex involved in the trunsduction of antiproliferative and cell adhesion signals. Expression level of IFITM1 was found significantly different between the high- and low-risk groups (P = 9.7976 x 10(-11)) by real-time reverse transcription polymerase chain reaction (RT-PCR). Higher IFITM1 expression correlated with improved survival (P = 0.01). These results indicate that IFITM1 expression profiling could be used for molecular classification of CML, which may also predict survival.

p38 Mitogen-activated protein kinase-dependent and -independent signaling of mRNA stability of AU-rich element-containing transcripts.

Adenylate/uridylate-rich element (ARE)-mediated mRNA turnover is an important regulatory component of gene expression for innate and specific immunity, in the hematopoietic system, in cellular growth regulation, and for many other cellular processes. This diversity is reflected in the distribution of AREs in the human genome, which we have established as a database of more than 900 ARE-containing genes that may utilize AREs as a means of controlling cellular mRNA levels. The p38 mitogen-activated protein kinase (MAP kinase) pathway has been implicated in regulating the stability of nine ARE-containing transcripts. Here we explored the entire spectrum of ARE-containing genes for p38-dependent regulation of ARE-mediated mRNA turnover with a custom cDNA array containing probes for 950 ARE mRNAs. The human monocytic cell line THP-1 treated with lipopolysaccharide (LPS) was used as a reproducible cellular model system that allowed us to precisely control the conditions of mRNA induction and decay in the absence and presence of the p38 inhibitor SB203580. This approach allowed us to establish an LPS-induced ARE mRNA expression profile in human monocytes and determine the half-lives of 470 AU-rich mRNAs. Most importantly, we identified 42 AU-rich genes, previously unrecognized, that show p38-dependent mRNA stabilization. In addition to a number of cytokines, several interesting novel AU-rich transcripts likely to play a role in macrophage activation by LPS exhibited p38-dependent transcript stabilization, including macrophage-specific colony-stimulating factor 1, carbonic anhydrase 2, Bcl2, Bcl2-like 2, and nuclear factor erythroid 2-like 2. Finally, the identification of the p38-dependent upstream activator MAP kinase kinase 6 as a member of this group identifies a positive feedback loop regulating macrophage signaling via p38 MAP kinase-dependent transcript stabilization.

Heterogeneity in control of mRNA stability by AU-rich elements.

AU-rich elements (AREs), located in the 3'-untranslated region of unstable cytokine and chemokine mRNAs, promote rapid decay of otherwise stable mRNAs and may mediate selective mRNA stabilization in response to stimulation with interleukin-1 (IL-1). AREs vary considerably, however, in both size and sequence context. To assess the heterogeneity involved in control of mRNA stability by ARE motifs, human mRNA sequences from IL-1alpha-stimulated HEK293 cells and T98G cells were screened for either instability or stability using both cDNA (950 ARE containing sequences) and Affymetrix oligonucleotide (U95Av2 GeneChip) array analysis. Although ARE-containing mRNAs exhibited a broad range of stability, IL-1alpha promoted stability in a subset of mRNAs that were unstable when transcriptionally induced by tumor necrosis factor alpha. Stabilization of granulocyte/macrophage-colony stimulating factor and IL-8 mRNAs by IL-1alpha was achieved only after 2 h of stimulation, required ongoing protein synthesis, and depended on the activation of p38 MAPK. In contrast, stabilization of Gro3 mRNA in response to IL-1alpha was achieved immediately and was insensitive to inhibitors of protein synthesis and p38 MAPK activation. In concert, these findings demonstrate that ARE sequences are functionally heterogeneous; only a subset of unstable mRNAs is sensitive to stabilization by IL-1alpha. Moreover, IL-1alpha promotes stabilization of unstable mRNAs through distinct mechanistic pathways that distinguish between specific mRNA sequences.

Thrombomodulin RNA is destabilized through its 3'-untranslated element in cells exposed to IFN-gamma.

Interferon-gamma (IFN-gamma) is a potent activator of mononuclear phagocytes, allowing them to play a prominent role in acute and chronic inflammatory responses. IFN-gamma binding to its cell surface receptor initiates changes in the steady-state levels of cellular RNAs, permitting the proteins encoded by these RNAs to exert its biologic actions. Hundreds of cellular RNAs have been identified whose rates of transcription are altered by incubation of cells with IFNs. The rates of transcription of many of the genes encoding these RNAs are enhanced by IFN-gamma-mediated activation of the Stat1 transcription factor that is tyrosine phosphorylated and translocates to the nucleus, where it binds enhancers present in IFN-stimulated genes (ISGs). IFN-gamma can also modify the concentrations of some RNAs by posttranscriptional mechanisms. However, very little is understood about the molecular mechanisms regulating this phenomenon. We have identified the RNA encoding thrombomodulin (TM), a physiologic receptor for thrombin, that is downregulated in primary human monocytes incubated with IFN-gamma. Using actinomycin D as a transcriptional inhibitor, we show that the mRNA half-life is rapidly shortened by IFN-gamma. The TM transcript contains a large 3'-untranslated region (UTR), with several AU-rich elements (AREs), elements that have been implicated in the regulation of mRNA decay. Using a tetracycline-regulatory promoter system, we analyzed RNA levels in the absence of transcription of TM. Results from these experiments indicate that incubation of cells with IFN-gamma accelerates the decay of TM RNA through its 3'-UTR. This is the first report describing a clear posttranscriptional downregulation of an mRNA by IFN-gamma that identifies the 3'-UTR as a target of IFN-gamma-stimulated destabilization.